JPS6141556B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a red pigment by culturing callus derived from plant tissue, and more specifically, by culturing callus derived from plant tissue of Hanakirin, a plant of the genus Euphorbia, in a medium to produce a red pigment chrysanthemine (cyanidin). -3-glucoside)
Concerning how to produce. Pigments are often used as food coloring agents to add aesthetic appeal to foods, but synthetic colorants pose various problems due to their toxicity, such as mutagenicity and carcinogenicity. . Therefore,
From the viewpoint of safety, it is desired to use pigments derived from natural products as coloring agents for foods and the like. However, natural cultivation is subject to constraints from the natural environment such as season, climate, temperature, latitude, etc.
It is not possible to maintain a stable supply by collecting from natural plants. In addition, large-scale cultivation using arable land naturally competes with food production, so there is a limit to its supply, and there is also a limit to its productivity, making it extremely expensive. However, in recent years, research on plant cell culture has been progressing as a method for producing plant components. Plant cell culture grows at a much faster rate than natural plants, which grow on a yearly or monthly basis, so it is possible to produce the desired ingredients in a short period of time, and unlike natural cultivation, it is less susceptible to the effects of weather etc. It has the advantage of being able to be produced in a planned manner on an industrial scale without the need for much labor for collection. Several methods have already been proposed for producing natural pigments using such cell culture methods. For example, in Japanese Patent Application Laid-open No. 49-94897, an anthocyanin-based pigment M-composed of multiple components obtained by tissue culture of Red-breasted wrinkles.
1 production method (yield 0.28%) is disclosed,
JP-A No. 53-84026 discloses a method for producing anthocyanin pigments consisting of multiple components by tissue culturing Ai plants, and JP-A No. 54-11281 discloses a method for producing anthocyanin pigments consisting of multiple components by tissue culturing plants belonging to the genus Delis. A method (yield 0.2%) for producing an anthocyanic pigment consisting of multiple components is disclosed. From this point of view, the present inventors have conducted intensive research to enable the industrial production of natural pigments by culturing plant cells, and as a result, we have developed a callus derived from the plant tissue of Hanakirin, a plant of the genus Euphorbia. The present inventors have discovered that the natural pigment chrysanthemine can be selectively produced in high yield by culturing, leading to the present invention. The red pigment produced by the present invention is chrysanthemine (cyanidin-3-glucoside) having the following structural formula, and since it is not a multi-component pigment but only one type of chrysanthemine, extraction and purification operations for the produced pigment are not possible. It's easy. The cultured plant cells used in the present invention are obtained by deriving Hanakirin, a plant of the genus Euphorbia, according to a conventional method. An example will be explained below. First, after thoroughly washing the leaves of Hanakirin with deionized water, they were immersed in 70% ethyl alcohol for 5 to 10 minutes, then in a 10% bleaching powder solution for 5 to 10 minutes to sterilize the bacteria attached to the surface. Wash away residual disinfectant with sterile distilled water. Next, the sterilized leaves are cut into small pieces with a sterile scalpel to an appropriate size, and placed on a synthetic medium containing an auxin agent such as 2,4-dichlorophenoxyacetic acid (for example, Murashige-Skoog medium). do. After placing on the bed, culturing is carried out in a light place under constant temperature conditions of 20 to 30°C, preferably around 28°C, preferably under light irradiation of 100 lux or more, more preferably 3000 to 100000 lux. After one week of such culture, a callus that produces the red pigment is formed from the leaves of Hanakirin, which is then transplanted onto a new agar medium of an appropriate composition and incubated at 20 to 30°C, preferably at 28°C. Under constant temperature, in a bright place, preferably 100 lux or more, more preferably 3000~
Continue culturing under light irradiation of 100,000 lux. In order to obtain callus on an industrial scale, the callus may be cultured and propagated by a static culture method or a liquid culture method in the same manner as in the culture of general microorganisms. As a liquid culture method, for example, a shaking culture method in which culture is performed on a shaking culture machine, or a method in which sterile air is aerated in a closed tank made of glass, metal, etc. may be selected as appropriate depending on the purpose. I can do it. The medium used for inducing or culturing the callus according to the present invention is based on various known inorganic synthetic agar media, and is supplemented with trace amounts of organic matter such as vitamins, carbon sources, plant hormones, and various naturally extracted substances. Use the added one. Representative examples of the inorganic synthetic agar medium include White medium, Hildebrand medium, and Rissmeyer medium.
Examples include Skoog medium and Murashige-Skoog medium. In addition, these media with improved compositions can also be used. Examples of trace organic substances such as vitamins include vitamins such as thiamine hydrochloride, pyridoxine hydrochloride, and nicotinic acid; amino acids such as glycine and asparagine; and hexahydric alcohols such as inosite and sorbitol. It may show good growth even without being added to the medium. As the carbon source, carbohydrates such as sucrose, glucose, and maltose; organic acids such as acetic acid; and alcohols such as methanol and glycerin can be used. Among them, sucrose is particularly preferred because of its high pigment productivity. The concentration used is 1-10% w/v, preferably 4-7% w/v. Examples of the plant hormones include auxin-acting substances such as 2,4-dichlorophenoxyacetic acid (2,4-D), β-indoleacetic acid (IAA), and α-naphthaleneacetic acid (NAA), and cytokinins such as kinetin. However, from the viewpoint of productivity and selectivity of the red pigment chrysanthemin, it is preferable to use 2.4-D for callus induction or culture. However, this does not preclude the use of other plant hormones other than auxin. The concentration of 2.4-D used is 10 ^-4 to 10 ^-7 M, preferably 10 ^-6 to
10 ^-7 M, and if this concentration is too high, callus will not grow, which is undesirable; if it is too low, callus will become edgy or not grow, which is undesirable. Examples of the various natural extracts include casein hydrolyzate (0-2% w/v), coconut milk (0-25% w/v), and yeast extract (0-2% w/v).
v), malt extract (0 to 2% w/v), etc. can be used alone or in any combination. In the method of the present invention, as described above, callus is cultured by placing a culture vessel containing callus under light irradiation of 100 lux or more, preferably 3,000 to 100,000 lux. As a light source, sunlight, a fluorescent lamp, an incandescent lamp, a mercury lamp, etc. can be used. If no light irradiation is performed, no dye will be produced. In order to separate and collect the red pigment chrysanthemin from the callus cultured in this way, known methods are used.
For example, it can be carried out by a solvent extraction method.
An example will be explained below. First, cells are cultured in a solvent containing an acid such as hydrochloric acid, acetic acid, or formic acid at a concentration of about 0.01 to 5% by weight, preferably water or an alcoholic solvent such as methanol or ethanol, and the culture is preferably lyophilized according to a conventional method. The cells are immersed at a temperature of -5 to 10°C and the produced red pigment is extracted. Next, the obtained extract was heated at 40°C after removing the solid content by filtration or other operations.
Concentrate under reduced pressure at the following temperature (too high a temperature is undesirable as the red pigment will easily decompose), transfer this concentrated solution to a separating funnel, and use ether (hexane, heptane, petroleum ether, etc. instead of ether). (Chloroform, methylene dioxide, ethyl acetate, etc. can also be used) and shake to dissolve oil-soluble impurities in ether and separate and remove them. After repeating this ether washing operation several times, the desired red pigment chrysanthemine can be obtained by drying the pigment extract under reduced pressure.
It can be purified by cellulose column chromatography, paper chromatography, etc. The obtained chrysanthemine can be identified by comparing it with standard chrysanthemine using paper chromatography or UV absorption spectrum, as shown in the following examples. As explained above, according to the method of the present invention, the natural red pigment chrysanthemin, which is suitable as a food coloring agent, can be produced in pure form by culturing callus derived from the plant tissue of Hanakirin, a plant of the genus Euphorbia, in a specific medium. can be produced in
It can be produced extremely efficiently compared to production from natural plants. Examples of the present invention will be described below, but it goes without saying that the scope of the present invention is not limited to the following examples. Example The leaves of Hanakirin were thoroughly washed with water and cut into approximately 4 cm ² pieces. Next, these sections were sterilized by immersing them in 70% ethyl alcohol for 5 minutes, then in a 10% bleaching powder solution for 10 minutes, and then washed by immersing them several times in sterile distilled water in a sterile box and thoroughly sterilizing them. Removed residual disinfectant. These leaf sections were cut into small pieces with a width of about 1 cm ² using a sterile scalpel, and the obtained small pieces of Hanakirin leaves were placed aseptically on a synthetic agar medium having the following composition. The medium was Murashige-Skoog's inorganic salt medium, 3% w/v of sucrose, 0.2% w/v of malt extract,
2.4-D10 ^-6 M, thiamine hydrochloride 0.1 ppm, pyridoxine hydrochloride 0.5 ppm, nicotinic acid 0.5 ppm, glycine 2 ppm and inositol 100 ppm were added to adjust the pH to 6.0, and agar 0.8% w/v was added and the mixture was kept constant. A medium sterilized according to the regulations was used. Small pieces of Hanakirin leaves placed on such a medium were cultured at a culture temperature of 25°C under light irradiation of 3000 lux. After one week, a red callus appeared around the cut end of the leaf. After one month, the callus that had grown large was divided into small pieces, transplanted aseptically into a medium with the same composition as that used for callus induction above, and cultured at a culture temperature of 25°C under light irradiation of 3000 lux. The same operation was repeated every 4 to 6 weeks for a total of 6 months. The cultured Hanakirin cells grown in this manner were separated from the solid medium, and water was removed in a vacuum freeze dryer to obtain 8.1 g of dried callus. This dried callus was ground in a mortar and then immersed in 2% hydrochloric methanol in a cold place for 24 hours. After filtration, methanol is removed from the extract obtained at a temperature below 40℃.
After concentrating to about 50ml and transferring to a separating funnel,
ml of ether was added, and after thorough shaking, the ether layer was separated. After repeating this ether washing operation several times, the mixture was further concentrated under reduced pressure at 40° C. or lower to dryness. This dried product was dissolved in a small amount of 0.01% hydrochloric acidic methanol, applied in a strip on cellulose TLC (20 cm x 20 cm), and developed using a developing solvent of hydrochloric acid/acetic acid/water = 5/1/5. The red band was scraped off and immersed in 2% methanol with hydrochloric acid to extract the red pigment. This extract was dried under reduced pressure at 40° C. or below to obtain 58 mg (yield 0.7%) of a black-red powder. Paper chromatography of this dye (Toyoro paper)
No. 51, same hereafter) (Solvent: n-butanol/acetic acid/water = 4/1/5 upper layer, n-butanol/hydrochloric acid/water = 5/1/4 upper layer, 1% hydrochloric acid and acetic acid/
As shown in Table 1 below, the R _f value of hydrochloric acid/water = 15/3/82) was in good agreement with that of standard chrysanthemin (extracted and isolated from Delaware grapes and structure determined).

[Table] Also, the obtained pigment and standard chrysanthemin
When we measured the UV absorption spectrum, we found that Figure 1A
As shown in and B, the UV absorption spectrum of the red dye was in good agreement with that of standard chrysanthemin. Next, the red pigment obtained above was boiled in 20% hydrochloric acid for 5 minutes, and the aglycone obtained by hydrolysis was subjected to paper chromatography (solvent: n-butanol/acetic acid/water = 4/
1/5 upper layer, n-butanol/hydrochloric acid/water = 5/
The R _f values of 1/4 upper layer, acetic acid/hydrochloric acid/water = 5/1/5 and formic acid/hydrochloric acid/water = 2/1/2) are also shown in Table 2 below. The results were in good agreement with those obtained by hydrolysis.

[Table] Furthermore, the results of paper chromatography (solvent: phenol/water/=4/1 and pyridine/butanol/water=3/6/1) of the sugar part of the red pigment are also shown in Table 3 below. Agreed. From these results, it can be identified that the red pigment obtained above is chrysanthemine.

【table】 [Brief explanation of the drawing]

Figure 1 is a chart of the UV absorption spectra of the red pigment obtained from callus in the example and standard chrysanthemin. Figure A shows the absorption spectrum of the red pigment of callus, and Figure B shows the absorption spectrum of standard chrysanthemin. show.

Claims (1)

[Scope of Claims] 1. A method for producing a red pigment, chrysanthemin, which comprises producing a red pigment, chrysanthemin, by culturing callus derived from the plant tissue of Hanakirin, a plant belonging to the genus Euphorbia. 2. The method according to claim 1, wherein the callus is cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at a concentration of 10 ^-4 to ^{10 -7} M. . 3. The method according to claim 1, wherein the callus is cultured under light irradiation of 100 lux or more. 4 The callus was cultured with 1 to 10% by weight of sucrose.
The method according to claim 1 or 2, which is carried out in a medium containing a concentration of .